The neurotoxicity of Semen Strychni has been reported recently in several clinical cases. Therefore, this study was conducted to investigate the role of HMGB1 in a model of neurotoxicity induced by Semen Strychni and to assess the potential alleviating effects of glycyrrhizic acid (GA), which is associated with the regulation of HMGB1 release. Forty-eight SD rats were intraperitoneally injected with Semen Strychni extract (175 mg/kg), followed by oral administration of GA (50 mg/kg) for four days. After treatment of SS and GA, neuronal degeneration, apoptosis, and necrosis were observed via histopathological examination. Inflammatory cytokines (TNF-α and IL-1β), neurotransmitter associated enzymes (MAO and AChE), serum HMGB1, nuclear and cytoplasmic HMGB1/ph-HMGB1, and the interaction between PP2A, PKC, and HMGB1 were evaluated. The influence of the MAPK pathway was also examined. As a result, this neurotoxicity was characterized by neuronal degeneration and apoptosis, the induction of pro-inflammatory cytokines, and a reduction in neurotransmitter-metabolizing enzymes. In contrast, GA treatment significantly ameliorated the abovementioned effects and alleviated nerve injury. Furthermore, Semen Strychni promoted HMGB1 phosphorylation and its translocation between the nucleus and cytoplasm, thereby activating the NF-κB and MAPK pathways, initiating various inflammatory responses. Our experiments demonstrated that GA could partially reverse these effects. In summary, GA acid alleviated Semen Strychni-induced neurotoxicity, possibly by inhibiting HMGB1 phosphorylation and preventing its release from the cell.

BACKGROUND: This paper aimed to establish a method to help investigate the combination mechanism of traditional Chinese medicine from the metabolic perspective.
BACKGROUND: Semen Strychni has been a frequently used herb in clinics for a long time. In traditional Chinese medical science, Semen Strychni always combinate with other herbs to modulate its nature of severe toxicity. However, the mechanism of the combination is still unclear. Previous research mostly focused on the components of the herbs. The metabolic processes of the main components of the Semen Strychni are also very important and have rarely been reported.
OBJECTIVE: This study tended to develop a rapid and sensitive high-performance liquid chromatographic- tandem mass spectrometric (HPLC-MS/MS) method for the determination of two major metabolites of Semen Strychni in rat urine.
METHODS: Chromatographic separation was carried out on a C18 column. Detection was performed by a selective reaction monitoring (SRM) mode via an electrospray ionization (ESI) source operating in the positive ionization mode. Analysis of analytes from rat plasma was carried out by protein precipitation using acetonitrile.
RESULTS: The assay was validated in terms of specificity, precision, recovery, etc. The intra- and inter-day precision values were less than 13.1%. The recoveries at three levels were more than 69.1%. The method was then used to study the kinetic profiles of the metabolites of Semen Strychni in rat urine for the first time.
CONCLUSIONS: The results demonstrate that the established LC/MS method in this study is accurate and sensitive for the simultaneous determination of the two metabolites of Semen Strychni in rats' urine samples. This method could be a supplement to the plasma pharmacokinetics of Semen Strychni.

Semen Strychni has long been used for the treatment of rheumatoid arthritis, facioplegia and myasthenia gravis due to its anti-inflammation and anti-nociceptive properties in China. However, the fatal neurotoxicity of Semen Strychni has limited its wider clinical application. To investigate the acute toxicity induced by Semen Strychni and the detoxification of liquorice, we evaluated inflammation, oxidative stress and the translocation of high mobility group box 1 (HMGB1) in rats. As a result, there were obvious oxidative stress and inflammation in hippocampus after the Semen Strychni extracts (STR) treatment in rats. Liquorice extracts (LE) and its three active monomers - glycyrrhizic acid (GA), liquiritigenin (LIQ), isoliquiritigenin (ISL) showed the potential for mitigating STR-induced neurotoxicity. HMGB1 levels in cytoplasm and serum and the levels of two downstream receptors RAGE and TLR4 were significantly increased after STR treatment. Through using LE and the monomers, the nucleocytoplasmic transport and release of HMGB1 were inhibited. In addition, the binding between HMGB1 and TLR4 was weakened in detoxification groups comparing with the STR group. Taken together, these findings indicated that liquorice and its active components alleviated acute neurotoxicity induced by Semen Strychni partly via HMGB1-related pathway.

To evaluate the detoxification effect of a combination of Radix Glycyrrhizae (GU) and Semen Strychni (SN) from toxicokinetics and drug tissue distribution perspectives, decoctions of processed SN and codecoction of SN and GU (SGN) were prepared, and an HPLC-ESI-MS/MS method was developed to monitor the severe exposure level in 1-month toxicokinetics and tissue distribution experiments to detect brucine and strychnine in rats. The toxicokinetic characteristics and tissue distribution before and after the addition of GU were analyzed. The method was successfully applied to evaluate the toxicokinetics and tissue distribution before and after the combination of SN and GU. The results show that GU decreased the blood concentration of toxic components in SN, and a double peak was observed in the drug time curve. The results of tissue distribution show that a combination of GU and SN significantly decreased the accumulation of toxic substances in metabolic organs and accelerated the clearance of toxic substances in the brain. These results provide a reference for the toxicity reduction mechanism of GU combined with SN.

Acute neurotoxicity of Semen Strychni can result in sudden death in epilepsy. The detoxification method and mechanism of Semen Strychni acute poisoning have not been clarified. This experiment focused on the mechanism of Semen Strychni neurotoxicity and the alleviation effects of isoliquiritigenin. The rats were intraperitoneally injected with Semen Strychni extract (125 mg/kg), followed by oral administration of isoliquiritigenin (50 mg/kg) for 7 days. FJ-B staining was used to evaluate the degree of injury on hippocampus neurons. The concentration of monoamines, amino acids, and choline neurotransmitters, the Dopamine (DA) and 5-hydroxytryptamine (5-HT) metabolic pathway in the hippocampus, cerebellum, striatum, prefrontal cortex, hypothalamus, serum, and plasma were detected by LC-MS/MS. The expression of neurotransmitter metabolic enzymes [catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO)] and neurotransmitter receptors [glutamate N-methyl-D-aspartic acid receptors (NMDARs) and gamma-aminobutyric acid type A receptor (GABRs)] were, respectively determined using ELISA and qRT-PCR. The results indicated that Semen Strychni induced neuronal degeneration in the hippocampal CA1 region. Meanwhile, Semen Strychni inhibited the mRNA expression of NMDAR1, NMDAR2A, NMDAR2B, GABRa1, GABRb2 and reduced the level of MAO, which disrupted the DA and 5-HT metabolic pathway. However, isoliquiritigenin reversed these effects. In summary, isoliquiritigenin showed alleviation effects on Semen Strychni-induced neurotoxicity, which could be attributed to restoring neurotransmitters metabolic pathway, most likely through the activation of NMDA receptors.

Rheumatoid arthritis is a systemic autoimmune disease characterized by synovial inflammation and bone destruction. Identifying drugs with time-varying efficacy and toxicity, and elucidating the mechanisms would help to improve treatment efficacy and reduce adverse effects. Here, we aimed to determine the chronoefficacy of semen strychni (SS) and tripterygium glycoside tablet (TGT) against rheumatoid arthritis in mice, and to investigate a potential role of circadian pharmacokinetics in generating chronoefficacy. SS extract and TGT suspension were prepared with ultrasonication. Effects of SS and TGT on collagen-induced arthritis (CIA) were evaluated by measuring TNF-α and IL-6 levels. SS dosed at ZT18 was more effective in protecting against CIA than drug dosed at ZT6 (i.e., lower levels of key inflammatory factors at ZT18 than at ZT6). This was accompanied by higher systemic exposure levels of strychnine and brucine (two main putative active ingredients of SS) in ZT18-treated than in ZT6-treated CIA mice. TGT dosing at ZT2 showed a better efficacy against CIA as compared to herb doing at ZT14. Consistently, ZT2 dosing generated a higher exposure of triptolide (a main putative active ingredient of TGT) as compared to ZT14 dosing in CIA mice. Moreover, strychnine, brucine, and triptolide significantly inhibited the proliferation of fibroblast-like synoviocytes, and reduced the production of TNF-α and IL-6 and the mRNAs of TNF-α, IL-6, COX-2, and iNOS, suggesting that they possessed an anti-arthritis activity. In conclusion, SS and TGT display chronoefficacy against rheumatoid arthritis in mice, that is attributed to circadian pharmacokinetics of main active ingredients. Our findings have implications for improving treatment outcomes of SS and TGT via timed delivery.

Strychnos alkaloids (SAs) are the main toxic constituents in Semen Strychni, a traditional Chinese medicine, which is known for its fatal neurotoxicity. Hence, the present study was carried out to evaluate the neurotoxicity induced by SAs and the pre-protective effects of the total glucosides of Paeoniae Radix Alba (TGP). An SA brain damage model was firstly established. The neurotoxicity induced by SAs and the pre-protective effects of TGP were confirmed by physical and behavioral testing, biochemical assay, and histological examination. Then, a liquid chromatography-tandem mass spectrometry method was developed and validated to investigate the time-course change and distribution of strychnine and brucine (two main SAs) in the brain after oral SA administration with or without TGP pretreatment. Biochemical analysis results indicated that TGP could ameliorate the oxidative stress status caused by SAs. Time-course change and distribution studies demonstrated that strychnine and brucine were rapidly absorbed into the brain, peaked early at 0.5 h, and were mainly located in the hippocampus and cerebellum. TGP showed a pre-protective effect against neurotoxicity by reducing the absorption of toxic alkaloids into the brain. These findings could provide beneficial information in facilitating future studies of Semen Strychni neurotoxicity and developing herbal medicines to alleviate neurotoxicity in the clinic.

In this study, we investigated the protective effect of total glycosides of paeony against Semen Strychni-induced neurotoxicity and discussed some probably mechanisms. Levels of estrone, estradiol, estriol and growth hormone in male rats\' serum were determined by ELISA, levels of ATP and substances associated with energy metabolism in rats\' brain were determined by HPLC and levels of progesterone was determined by a UPLC-MS/MS method. The results showed that neurotoxicity induced by Semen Strychni could cause a significant decrease (p < 0.05, compare to the blank group) in secretion of estrogens and GH and disorder brain energy metabolism at the same time. While, rats with total glycosides of paeony pre-protection (orally administrated with total glycosides of paeony for 15 days before administrating Semen Strychni extract) showed a much better condition in the secretion of hormones and brain energy metabolism, and showed no significant changes in most of those associated substances when comparing to the blank group. Our study indicated that total glycosides of paeony have neuroprotective effects on Semen Strychni-induced neurotoxicity. It could recover the disordered hormone secretion and improve the brain energy metabolism. Total glycosides of paeony is potential to be further used in clinic to protect against neurotoxicity induced by other reasons.

Semen Strychni is known for its treatment of rheumatic arthritis with a low therapeutic index. Liquorice contributes a lot in herb detoxification according to the traditional Chinese medicine theory. A simple, rapid, and sensitive liquid chromatography-mass spectrometric method (LC-MS) was developed and validated for simultaneous determination of main bioactive ingredients in liquorice and Semen Strychni in rat plasma. Using moclobemide and cyproterone acetate as the internal standards, the analytes were pretreated via protein precipitation with methanol. An Ultimate AQ-C18 column (3.0 μm, 3.0 × 100 mm) was employed for chromatographic separation, combining with gradient elution. The mobile phase consisted of 0.07% formic acid and 0.12% ammonium acetate in aqueous phase (A) and acetonitrile in organic phase (B). The elution program was as follows: 0-0.5 min, 20% B; 0.5-1 min, 20-60% B; 1-7 min, 60-85% B; and 7-7.5 min, returned to 20% B, then continued to 12 min. Selected reaction monitoring was performed in both positive and negative ESI. Positive mode was adopted for detection of strychnine, brucine, and moclobemide, while negative mode was used for glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin, isoliquiritigenin, liquiritin, and cyproterone acetate. The method was validated for specificity, linearity, matrix effect, recovery, precision, accuracy, and stability. The results show that this method is sensitive, accurate and robust for biological matrix analysis. Moreover, the proposed method was applied to a pharmacokinetic study in Sprague-Dawley rats for investigating the mechanism of which liquorice detoxifies Semen Strychni.

Semen Strychni has been widely used as a traditional Chinese herb medicine, but its clinical use was limited for its potential neurotoxicity and nephrotoxicity. This study aimed to investigate S. Strychni-induced neurotoxicity and the neuro-protective effect of Paeonia lactiflora based on monitoring nine potential neurotoxicity biomarkers in rat serum and brain tissue. A sensitive liquid chromatography-tandem mass spectrometry method was developed and validated to monitor serotonin, tryptophan, dopamine, tyrosine and glutamate in serum and five brain regions (prefrontal cortex, hippocampus, striatum, cerebellum and hypothalamus). Analytes were separated on a CAPCELL CORE PC column (150 mm × 2 mm, 2.7 μm) with a gradient program of acetonitrile-water (0.2 % formic acid) and a total runtime of 7.5 min. In addition, enzyme-linked immunosorbent assay was conducted to determine four kinds of protein (tryptophan hydroxylase, tyrosine hydroxylase, endogenous brain-derived neurotrophic factor and nerve growth factor). Results demonstrated that the administration of S. Strychni could cause certain endogenous substances disorder. These analytes were found significantly changed (p < 0.05) in serum (except glutamate) and in certain tested brain regions in S. Strychni extract group. Pretreatment of P. lactiflora could significantly reverse the S. Strychni-induced neurotoxicity and normalize the levels of such endogenous substances. The study could be further used in predicting and monitoring neurotoxicity caused by other reasons, and it was expected to be useful for improving clinical use of S. Strychni through pretreatment with P. lactiflora.